Biomedical Engineering Reference
In-Depth Information
Table 4.2
Techniques to study the parameters influencing the structure and the composition of
the protein corona
Corona
parameter
Impacts on the nano-systems
Techniques
Thickness and
density
Influence on the hydrodynamic size of the
nanomaterial
DLS, DCS, SEC, TEM
Identity and
quantity
Influence on the array of biological interactions PAGE, LC-MS/MS
Conformation
Influence on the activity of a protein and its
interaction
CD, fluorescence quenching,
computational simulation
Affinity
Influence on the biophysical interactions or
translocation to a new physiological
compartment
SEC, SPR, ITC
DLS
dynamic light scattering,
DCS
differential centrifugal sedimentation,
SEC
size-exclusion
chromatography,
TEM
transmission electron microscopy,
PAGE
polyacrylamide gel electropho-
resis,
LC-MS/MS
liquid chromatography-mass spectrometry,
CD
circular dichroism,
SPR
surface
plasmon resonance,
ITC
isothermal titration calorimetry
time (often between 10 min and a few hours) and washing the unbound proteins
using ultracentrifugation [
5
], column chromatography [
6
], or density gradient
purification [
7
]. It can be safely assumed that 3-4 centrifugation washes can get
rid of most of the plasma protein. However, there is a risk of losing weakly bound
proteins.
The protein corona is identified by different parameters such as thickness,
density, protein identity, protein quantity, protein-NP affinity, protein arrangement,
and protein conformation. The importance of each parameter as well as the charac-
terization techniques for each of them is summarized in Table
4.2
.
Aggarwal et al. [
8
] summarized recent researches on the protein corona of
various nanoparticles as well as the employed techniques for protein isolation,
separation, and identification. The most conventional methods for protein isolation
are centrifugation, gel filtration, magnetic separation for magnetic nanoparticles,
and affinity chromatography. In the case of protein separation and identification, the
most applicable methods are 2D-PAGE and SDS-PAGE.
In this chapter the most conventional techniques for protein corona isolation,
separation, and identification are reviewed.
4.1 Centrifugation
Most studies of protein corona experiments start with incubation of nanoparticles
inside the blood plasma. Centrifugation is the most conventional method for
isolating nanoparticle-protein corona from the rest of plasma and loosely bound
proteins. There are some concerns about the centrifugation method which during
the analysis of the data should be considered. The washing duration, steps, and
